明胶海绵与人胚半月板细胞构建复合物的研究

目的：研究明胶海绵作为半月板组织工程支架材料应用的的可行性。方法：采用胶原酶阶段消化法获取人胚半月板纤维软骨细胞，取经胰岛素样生长因子I(IGF—Ⅰ)扩增的第3代细胞与已用多聚赖氨酸修饰的明胶海绵复合培养，进行MTT、光镜、扫描电镜观察。结果：纤维软骨细胞于明胶海绵上牢固粘附，4d开始增殖，3周时已有大量细胞生长于明胶海绵上。结论：明胶海绵表现出良好的亲水性、细胞吸附性和体外生物相容性，适于作为组织工程细胞培养支架材料用。     

兔髁突细胞与可吸收明胶海绵复合培养的研究

目的: 研究明胶海绵作为颞下颌关节组织工程支架材料应用的可行性.方法: 采用胶原酶阶段消化法获取兔髁突软骨中的纤维软骨细胞,取经碱性成纤维细胞生长因子(bFGF)扩增的第3代细胞与已用多聚赖氨酸修饰的明胶海绵复合培养,进行噻唑蓝(MTT)比色及光镜、扫描电镜观察.结果: 纤维软骨细胞于明胶海绵孔壁内牢固粘附,5 d开始增殖,4 W时有大量细胞生长于明胶海绵上.结论: 明胶海绵表现出良好的亲水性、细胞吸附性和体外生物相容性,可作为组织工程细胞培养支架材料用.     

探讨不同浓度 SPIO 对人成纤维细胞标记率和细胞活力影响及其MRI的实验研究

目的:探讨不同浓度超顺磁性氧化铁纳米粒子( Super-paramagnetic iron oxide,SPIO)体外标记人成纤维细胞 (Human fibroblast cells,Hfbs) 的磁性标记率、对细胞生长活力的影响,以及体外磁共振成像(Magnetic resonance imaging,MRI) 成像的特征.方法:将单纯 SPIO(铁浓度为 200 μg/ml )、不同浓度的SPIO-多聚赖氨酸复合物分别与培养基混合( 铁的终浓度分别为 150、100、50、25 μg/ml,PLL 终浓度为 7.5 μg/ml ),加入 Hfbs 共同培养 12、24、48、72 h 后收集细胞.采用台盼蓝排除试验检测细胞活性和普鲁士蓝染色法检测细胞铁分布.运用光学显微镜、透射电镜观察铁颗粒在细胞内的位置、计算其标记率;MR 扫描观察细胞信号强度变化情况.结果:SPIO-多聚赖氨酸复合物组细胞标记率显著高于单纯 SPIO 标记组 (P<0.05 ), 台盼蓝排除试验显示,100 μg Fe/ml 及其以下浓度组细胞活力与对照组之间差异无统计学意义(P>0.05 ).普鲁士蓝染色显示每个标记细胞胞质内可见多少不等的蓝染铁颗粒,电镜显示铁颗粒集中于内涵体和溶酶体中;含量在 25～50 μg Fe/ml 的培养液是 SPIO 标记干细胞的安全浓度,在此浓度阈值标记后孵育 18～24 h 即可有效标记细胞 95%～100% ;MR扫描显示标记细胞信号强度明显降低.结论:自制合成的 SPIO-多聚赖氨酸复合物可以简便、安全的标记人成纤维细胞.并且在适当浓度 25 μg Fe/ml 标记细胞不仅标记效率高,而且对细胞活力无明显影响,SPIO 标记细胞明显降低MR扫描下的细胞信号强度.     

三种人工合成可降解血管支架材料体外生物相容性及表面改性研究

目的:对聚对苯二甲酸丁二醇酯-co-聚对苯二甲酸环己烷二甲醇酯-b-聚乙二醇嵌段共聚物(T20)、聚3羟基丁酸-3-羟基戊酸酯(PHBV)和聚对苯二甲酸丁二醇酯/聚乙二醇对苯二甲酸酯嵌段共聚物(PEGT/PBT)共三种人工合成的可降解血管支架材料进行体外生物相容性评价,并观察它们表面改性后对犬血管平滑肌细胞黏附生长的影响.方法:通过测定接触角观察三种支架材料的亲水性;MTT法、流式细胞术(FITC)及扫描电镜观察犬血管平滑肌细胞在T20、PHBV和PEGT/PBT三种支架上的增殖及生长情况,评价它们的体外生物相容性;采用明胶及多聚赖氨酸对三者进行表面预处理,MTT法比较三者表面改性后犬血管平滑肌细胞的黏附生长情况.结果:三种材料的接触角均小于90°,亲水性的大小顺序为:PEGT/PBT＞T20＞PHBV.MTT法显示犬血管平滑肌细胞可以在PHBV、PEGT/PBT、F20支架材料上黏附生长,接种24 h后细胞活性升高(PEGT/PBT＞T20＞PHBV,P＜0.01),随后降低,72 h后又开始增高(T20＞PEGT/PBT＞PHBV,P＜0.01);FITC荧光染色可见犬血管平滑肌细胞在载体上成片生长,状态良好;扫描电镜见犬血管平滑肌细胞在载体上呈片状融合生长,并见大量的细胞外基质.MTT法结果表明三种材料经过明胶或多聚赖氨酸表面改性预处理后细胞增殖活性明显高于未处理组(P＜0.01).结论:T20、PHBV、PEGT/PBT血管支架材料具有良好的体外生物相容性,对其进行表面改性后可明显提高犬血管平滑肌细胞的黏附生长能力.     

骨髓间充质干细胞体外诱导分化与标记

目的:探讨骨髓间充质干细胞(MSCs)在体外的诱导分化及5-溴脱氧尿嘧啶核苷(Brdu)标记MSCs的可行性. 方法:利用密度为1.073 kg/L的percoll分离骨髓的单个核细胞,体外培养与扩增MSCs;取生长良好的第3代细胞,用成骨细胞分化培养液培养21 d,Ⅰ型胶原免疫组化染色和碱性磷酸酶(AKP)组化染色;分别以浓度为5,10和15 μmol/L的Brdu溶液标记MSCs,孵育12,24,48,72和96 h后免疫组化检测Brdu,确定最佳标记量和最佳标记时间. 结果:诱导分化第21日, AKP染色呈强阳色,Ⅰ型胶原免疫染色呈阳性. 10 μmol/L Brdu标记MSCs的效果最好,随着孵育时间的延长,Brdu标记率逐渐增高,标记72 h后标记率在90%以上. 结论:MSCs在体外一定条件下可定向诱导分化为成骨细胞,用Brdu标记MSCs的最佳浓度为10 μmol/L,最佳时间是72 h.     

纤维蛋白支架对人嗅鞘细胞髓鞘形成相关蛋白表达的影响

目的:探讨纤维蛋白支架对人嗅鞘细胞髓鞘相关蛋白表达的影响.方法:将体外培养的成人鼻粘膜嗅鞘细胞种植于纤维蛋白支架,用多聚赖氨酸修饰的玻片作为对照底物,培养14 天后用扫描电镜观察细胞的形态学变化并对细胞内髓鞘形成相关蛋白2,3-环核苷3-磷酸二酯酶(2′,3′ cyclic nucleotide 3′ phosphodiesterase,CNPase)和髓鞘碱性蛋白(myelin basic protein, MBP)进行免疫荧光染色定位和免疫印迹半定量测定,分析纤维蛋白支架对人嗅鞘细胞CNPase和MBP表达的影响.结果:人嗅鞘细胞种植于纤维蛋白支架后,其形态为长条带状,细胞成束排列,方向一致.细胞内CNPase和MBP表达水平明显高于对照组.结论:纤维蛋白支架与人嗅鞘细胞具有很好的生物相容性并可促进细胞表达髓鞘相关蛋白CNPase和MBP;将该组织工程支架用于移植治疗脊髓损伤,有利于再生神经纤维的髓鞘形成.     

脱细胞骨骼肌支架与雪旺细胞构建体外人工神经模型的研究

目的：研究来源于自体骨骼肌组织的脱细胞细胞支架和自体雪旺细胞作为种子细胞的混合培养物作为一种神经桥接物的可能性。方法：将体外培养、扩增的雪旺细胞与用化学萃取制备且经过无菌多聚赖氨酸预处理的脱细胞骨骼肌支架进行混合培养2周,用扫描电子显微镜观察两者生长与附着情况。结果：雪旺细胞可与自体脱细胞骨骼肌支架艮好附着,并在支架上增殖、迁移,形成类似Bungner带的雪旺细胞链。结论：将自体脱细胞骨骼肌支架与雪旺细胞混合培养而构建体外人工神经模型是可行的。     

壳聚糖制作组织工程软骨支架的研究

目的:探讨在动物体内,利用新型生物材料壳聚糖制作软骨组织的可行性。方法:在体外,将培养的兔关节软骨细胞,种植于多聚赖氨酸包埋的壳聚糖支架上。体外培养4周,对组织工程软骨进行标本观察,HE染色,检测软骨内DNA含量。结果:制备的复合支架成形良好,交联后的机械强度明显提高。结论:壳聚糖适合细胞的贴附生长,可作为间充质干细胞的载体。     

血红素加氧酶-2基因敲除对Fe2+诱导的小鼠脑氧化应激损伤中基底节细胞的保护作用

目的:以人体骨髓间充质干细胞(BMSCs)为种子细胞,以去细胞猪主动脉瓣为支架,体外三维构建组织工程心脏瓣膜(TEHV).方法:以胸外科术中切除的肋骨为骨髓来源,采用1.073 g/ml的Percoll梯度分离液离心法获取BMSCs,体外培养扩增、鉴定;以0.05%胰酶 0.02?TA消化法制备去细胞猪主动脉瓣叶支架,将体外培养扩增的BMSCs种植于支架上,体外三维培养,应力环境下构建TEHV,观察TEHV细胞分泌的一氧化氮(NO)及内皮素(ET-1)水平、免疫组化特征及光镜和电镜下的结构.结果:人BMSCs为梭形,α-SMA及Vimentin染色阳性,CD-31及Ⅷ因子染色阴性,其分泌NO及ET-1分别为(104.1±5.9)μmol/L及(40.9±7.6)ng/L,明显较正常人血管内皮细胞低(P＜0.01);猪去细胞瓣膜支架去细胞完全,弹力纤维及胶原纤维保持完整;应力条件下体外三维构建的TEHV上活细胞数较静态下构建明显增多(P＜0.01),H-E染色示人BMSCs在支架上生长良好,形成完整的细胞层,扫描电镜示瓣膜表面细胞层完整,细胞呈梭形生长.结论:人BMSCs作为种子细胞已初步具有内皮细胞功能,在去细胞猪主动脉瓣膜支架上生长良好,可作为一种有前途的种子细胞构建自体TEHV;体外三维应力条件下构建TEHV效果明显优于静态培养.     

葡聚糖磁性纳米颗粒在基因转染中的应用

目的:评价葡聚糖磁性纳米颗粒作为基因载体在体外转染中的可行性。方法:用紫外分光光度计及琼脂糖凝胶电泳法分析检测葡聚糖磁性纳米颗粒结合DNA的能力,再将葡聚糖磁性纳米颗粒作为基因载体连接绿色荧光蛋白pEGFP-C1质粒报告基因在体外转染人膀胱肿瘤细胞BIU87和人树突状细胞,并在转染后72 h采用MTT比色法测定这种载体对细胞增殖和功能的影响以了解其细胞毒性。结果:在葡聚糖磁性纳米颗粒表面修饰多聚赖氨酸后,以静电引力作用结合DNA,其DNA结合率可达到75.6%,修饰多聚赖氨酸的葡聚糖磁性纳米颗粒可作为基因载体将报告基因转染至各类型细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞。与相同条件下脂质体为对照,转染后的细胞增殖活性及功能略优于对照组,有效地证明了葡聚糖磁性纳米颗粒的细胞毒性稍低于脂质体的细胞毒性。结论:葡聚糖磁性纳米颗粒可作为有效的基因载体之一。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.